Prospective Evaluation of the BD MAX StaphSR Assay for the Screening of Methicillin-Susceptible and -Resistant Staphylococcus aureus from Nasal Swabs Taken in Intensive Care Unit Patients

Screening patients for S. aureus nasal carriage has proved effective in preventing cross-contamination and endogenous infection with this bacterium. The aim of this study was to assess the performance of the BD MAX StaphSR assay with liquid Amies elution swabs, taken during routine care of intensive care unit patients. Direct and pre-enriched cultures were used as reference methods to screen for S. aureus and methicillin-resistant S. aureus (MRSA). Discrepant results between the BD MAX StaphSR assay and cultures were resolved by using the Xpert SA Nasal Complete assay. A total of 607 nasal swabs taken from 409 patients were included in this study. Compared to culture methods, the sensitivity and specificity of the BD MAX StaphSR assay were 92.5% and 91.7% for S. aureus screening, and 94.7% and 98.3% for MRSA screening, respectively. In 52 (8.6%) specimens, there was a discrepancy between the results of cultures and the BD MAX StaphSR assay, including 13 (25%) where the results of the BD MAX StaphSR assay were confirmed by the Xpert SA Nasal Complete test. This prospective study showed that the BD MAX StaphSR assay is reliable for S. aureus and MRSA detection from nasal samples taken with liquid Amies elution swabs.


Introduction
Staphylococcus aureus infections are a global public health concern as this species is the leading cause of bacterial-related mortality with more than 1 million deaths per year.In France, S. aureus is also the leading bacterial cause of death in 2019, surpassing Enterobacteriaceae [1].S. aureus is a common human commensal species colonizing around 24% of the general population [2].The vestibulum nasi is recognized as the main reservoir of S. aureus in humans, but the entire nasal cavity is also frequently colonized [3].S. aureus nasal carriers have an increased risk of endogenous infection with the S. aureus strain they carry.In most cases, the strain of colonization and the strain identified during the infection are genetically related [4,5].Screening and decolonization strategies are effective in preventing S. aureus surgical site infections and methicillin-resistant S. aureus (MRSA) transmission [6,7].
Screening based on culture methods including chromogenic agar plates is useful for the detection of S. aureus and MRSA, but it takes several days and has lower sensitivity than Nucleic Acid Amplification Tests (NAAT) [8].Third-generation NAAT, including the BD MAX StaphSR assay, detect S. aureus by amplifying a species-specific gene (e.g., the nuclease (nuc) gene or the staphylococcal protein A (spa) gene.These assays can distinguish methicillin-susceptible S. aureus (MSSA) from MRSA given the presence of both the methicillin-resistant gene (mecA/C) and the Staphylococcal Cassette Chromosome mec (SCCmec) embedded into the attB site of the S. aureus chromosome located at the 3 end of orfX.Several studies have highlighted the excellent performances of the fully automated BD Max StaphSR assay using positive blood cultures, wound swabs, and strains collections [9][10][11][12][13][14].Two other studies have evaluated the performances of this assay on nasal swabs for the detection of S. aureus and MRSA, but these studies were conducted in the United States and never in Europe, where the rate of nasal colonization by MRSA is lower than in the United States [15][16][17].
The aim of this study was to evaluate the BD MAX StaphSR assay for the detection of both MSSA and MRSA in nasal swabs taken in French intensive care units, using direct and pre-enriched cultures as the reference method.

Results
Six hundred and seven nasal Amies liquid elution swabs were included in the study.The BD MAX StaphSR assay was performed directly from the Amies liquid by transferring 200 µL into the sample buffer tube of the BD MAX StaphSR kit.In parallel, all swabs were tested by both direct and pre-enriched cultures on chromogenic agar plates to detect S. aureus and MRSA.Bacterial identification was confirmed by Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS).Susceptibility tests were performed by in-house PCR (mecA and mecC genes) and by cefoxitin disk diffusion test.Xpert SA Nasal Complete assays were used to analyze the discrepant results between the BD MAX StaphSR assays and culture methods.The results of culture methods combined with Xpert SA Nasal Complete assays permitted to define gold standard status for each sample.

Samples and Culture Methods
Based on the pre-enriched culture method, 27.9% [95% Confidence Interval (CI) (n = 19/159) of MRSA by using the pre-enriched culture method.Pre-enriched culture enabled to recover S. aureus in 14 additional samples, including one sample yielding MRSA which corresponded to an increase of sensitivity of 9.7% and 5.6% for the detection of S. aureus and MRSA, respectively.

Evaluation of BD MAX StaphSR Assay
The performances of the BD MAX StaphSR assay for the detection of S. aureus and MRSA in nasal swabs are depicted in Tables 1 and 2. The rate of inhibition was 0.7% [95% CI 0.2-1.7%] at the first attempt, and all samples had a valid result after retesting.
We first investigated the sensibility and specificity of the assay for S. aureus detection regardless of methicillin susceptibility.The BD MAX StaphSR assay detected S. aureus in 44 samples detected negative by the direct culture method (Tables 1 and A1).Of these samples, the pre-enriched culture recovered S. aureus in 7 samples, and 13 other samples were tested positive for S. aureus with the Xpert SA Nasal Complete assay.Conversely, pre-enriched culture recovered S. aureus in 12 samples that were not detected positive by the BD MAX StaphSR assay (Table A2).The Xpert SA Nasal Complete assay detected MSSA in 5 of these samples.In few cases, the pre-enriched culture method enabled to recover S. aureus in NAAT negative samples.In contrast, the BD MAX StaphSR assay detected S. aureus in 24 samples that were negative by culture and Xpert SA Nasal Complete assay (Table 1).The mean cycle threshold (CT) value (± SD) of nuc target for these samples was 33.1 ± 3.4, including five samples with CT > 37, which indicate a low S. aureus load.Next, we examined the performance of the BD MAX StaphSR assay for the detection of MRSA (Tables 2 and A1).All samples recovering MRSA by direct culture were detected positive for MRSA by the BD MAX StaphSR assay.One further sample was tested positive for MRSA by pre-enriched culture, but this sample was not detected as positive by either the BD MAX StaphSR nor the Xpert SA Nasal Complete assays (Table A2).The strain isolated from this sample was correctly identified as MRSA by the BD MAX StaphSR assay.On the other hand, seven culture-negative samples were found to be positive for MRSA by the BD MAX StaphSR assay.The targets used to define the presence of MRSA (i.e., spa, mecA, SCCmec) were amplified by the Xpert SA Nasal Complete assay in two samples, but only one sample was interpreted as positive by the GeneXpert Dx System software (version 2.1).In five of these samples, only the SCCmec-orfX right-extremity junction (MREJ) and mecA/C targets were detected on the BD MAX system.The difference between the CT values of both targets exceeded 2 CT for four of these samples.For the three remaining discrepant cases, the presence of MSSA was detected by culture and Xpert SA Nasal Complete assay.To further investigate these three cases, the BD MAX StaphSR assay was performed directly from the three S. aureus strains recovered by culture.In both cases, the S. aureus species-specific and the MREJ targets, but not the mecA/C target, were detected directly from the bacterial strains, which suggests that these strains were MRSA mecA dropout genotypes.These nasal swabs were suspected to contain a mixture of MRSA mecA dropout and methicillin-resistant coagulase-negative staphylococci (MR CoNS).Based on the sample status defined by the gold standard of this study, the sensitivity and specificity of BD MAX StaphSR assay for MRSA detection were 95.0% [95% CI 75.1-99.9%]and 98.5% [95% CI 97.1-99.3%],respectively (Table 2).

Discussion
S. aureus is still a leading cause of bacterial-related mortality and a major agent of nosocomial infections.This bacterium has developed important drug-resistance mechanisms over the decade [1,18].Bundled infection control programs involving screening for S. aureus and MRSA colonization by NAATs (at least one nasal swab plus one perineal or one throat swab) combined with decolonization have shown significant efficiency in reducing the spread and surgical site infections [1,6,7,19].These screening tests need to be repeated over time, as decolonization of the nose and other colonization sites is successful but short-lived [19,20].The use of effective screening methods is essential.In 2018, the World Health Organization recommended perioperative decolonization for all patients undergoing cardiothoracic and orthopaedic surgery (strong recommendations) or other types of surgery (conditional recommendations) with known nasal carriage of S. aureus [21].The implementation of a screening and decolonization strategy and an antimicrobial stewardship program [22] is crucial for reducing S. aureus infections, antibiotic consumption, length of hospital stay, and readmissions.
Our prospective study has shown that the BD MAX StaphSR assay is reliable for detecting both MSSA and MRSA colonization from nasal swabs in intensive care patients.Several studies were carried out to evaluate the BD MAX StaphSR assay for the detection of S. aureus and MRSA directly from samples for different indications.From positive blood cultures, the sensitivity and specificity were found to range from 99.4% to 100% and 99.5% to 100% for S. aureus detection, and from 97.9% to 100% and 98.1% to 100% for MRSA detection, respectively [9][10][11].It is worth noting that Dalpke et al. identified six S. aureus culture-negative bottles with a false-positive BD MAX StaphSR test result that were considered to be a bench-level contamination, thus overestimating our calculated range of specificity.Overall, the performance from positive blood cultures is better than in our study, which is probably explained by the high S. aureus load in positive blood cultures.Another study carried out on 250 wound swabs taken from US patients showed a sensitivity of 100% and 98.2% and a specificity of 95.2% and 99.5% for the detection of S. aureus and MRSA, respectively, which is slightly higher than the results observed in our cohort [12].Finally, only two published studies have evaluated the BD MAX StaphSR assay for the screening of nasal carriers.These two studies reported a sensitivity of 96.4% and 93.6% for S. aureus and MRSA detection, respectively, and a specificity of 93.1% and 97.7% for S. aureus and MRSA detection, respectively [15,16].As both studies were carried out in North America, the prevalence of MRSA nasal carriage was high due to the widespread of the community-acquired methicillin-resistant USA300 clone, and most MRSA isolates may belong to the USA300 lineage [15][16][17].In contrast, our study is the first to be carried out in Europe to evaluate the performance of the BD MAX StaphSR assay for MRSA and MSSA nasal carriage screening.Although the spreading of MRSA clones is strikingly different in Europe and North America, the BD MAX StaphSR assay showed excellent sensitivity and specificity to detect S. aureus and MRSA nasal carriers in our intensive care units.Moreover, the prevalence of S. aureus and MRSA nasal carriage was consistent with those observed in similar studies performed in Europe [2].
Although the results of our study were excellent, there are some limitations.As this study was monocentric in a cohort of intensive care patients, local epidemiology may have influenced the results.In addition, the SA Xpert Nasal Complete assay was performed only on specimens that gave discrepant results, which may lead to a bias in the definition of the gold standard status of the specimens.
Interestingly, we observed a few cases with controversial results between the techniques used in our study.Firstly, the BD MAX StaphSR assay detected MRSA in five culture-negative samples, while the nuc target was not detected.In fact, the BD Max StaphSR assay identifies MRSA when both MREJ and mecA/C are detected together, regardless of the result of the nuc target, with the aim of not missing MRSA strains [10].Ellem et al. and Van Leeuwen et al. reported strains having a divergent nuc gene which did not amplify with nuc primers but was femA-positive [10,23].Such strains have been recognized in the past, and the inability of this assay to amplify nuc has also been previously reported in MRSA strains [23], but this phenomenon seems to occur with a very low frequency worldwide.However, as the strains were not obtained by culture, it was not possible to decipher these false-positive MRSA cases.Secondly, of the three samples we suspected of containing a mixture of mecA dropout S. aureus and MR CoNS, one sample showed dissociated CT values between SCCmec and mecA/C targets, which suggests the presence of MR CoNS in this sample.Unfortunately, in the two remaining cases, the CT values of the targets were too close to suspect a mixture.Indeed, third-generation PCR assays targeting the SCCmec-orfX right-extremity junction and the mecA gene were found to reduce the occurrence of false-positive results due to mecA dropout, but they do not eliminate it in the case of patients colonized by both S. aureus-mecA dropout and the CoNS-carrying mecA gene.In a collection of strains isolated in the US, 7.1% of MSSA isolates were compatible with the genotype of the S. aureus empty-cassette variant [13].Lee et al. reported a prevalence of mecA dropout-S.aureus of 4.8% in atopic dermatitis samples from a Korean cohort [24].Unfortunately, we did not note the presence of Coagulase-Negative Staphylococci (CoNS) in culture.The prevalence of mecA dropout-S.aureus not being neglectable, the algorithms for interpreting NAAT still need to be refined to avoid false-positive results when these isolates are mixed with MR CoNS.Lastly, one MRSA culture-positive sample was missed by both BD MAX StaphSR and Xpert Nasal Complete assays.Although sequence variations in the SCCmec-orfX junction previously described lead to false-negative MRSA results in other NAATs [25,26], the false-negative result observed in our study was likely due to a very low S. aureus load in the sample, as the strain was correctly identified as MRSA by the BD MAX StaphSR assay.
Another study has shown that liquid Amies elution swabs are suitable to be used with the BD Max StaphSR assay [15].The excellent performance and the low level of inhibition observed in our cohort confirm that liquid Amies elution swabs can be used with the BD Max StaphSR assay in clinical practice.

Patients and Samples
A total of 607 nasal swabs (eSwab ref. 480 CE and 484 CE, Copan, Brescia, Italy) were included in this prospective study.Samples were collected consecutively without selection criteria during routine care from 409 intensive care patients, including children and adults, at the University Hospital of Saint-Etienne, France.All samples were tested by both the BD MAX StaphSR assay, direct culture, and pre-enriched culture.

BD MAX StaphSR Assay
Following the manufacturer's recommendations, swabs were vortexed briefly and a 200 µL volume of Amies liquid was added directly into the sample buffer tube provided with the kit.The BD MAX StaphSR assay has been designed to detect 3 targets including the nuc gene, the SCCmec-orfX right-extremity junction (MREJ), and the mecA/C gene.The result of the amplification of these 3 targets was automatically interpreted by the algorithm of the BD MAX system.

Culture Methods, Identification, and Susceptibility Tests
All samples were tested by both direct and pre-enriched cultures for MSSA and MRSA detection.For direct culture, swabs were streaked on chromogenic agar plates dedicated to the screening of S. aureus (BBL CHROMagar Staph aureus, Becton Dickinson, Le Pont de Claix, France) and MRSA (BBL CHROMagar MRSA II, Becton Dickinson).Agar plates from direct culture were incubated at 36 • C for 48 h.For pre-enriched culture, a 6.5% NaCl broth (BBL Salt Broth, modified, Becton Dickinson) was inoculated with 100 µL of sample and incubated at 36 • C overnight.If the broth was growing, a 10 µL volume of broth was streaked onto chromogenic agar plates (BBL CHROMagar Staph aureus and BBL CHROMagar MRSA II, Becton Dickinson) and incubated at 36 • C overnight.Presumptive colonies of S. aureus or MRSA were identified by MALDI-TOF MS (Microflex LT, Bruker Daltonics, Bremen, Germany).All S. aureus isolates on MRSA chromogenic agar plates were tested for methicillin resistance by using a cefoxitin susceptibility test and an in-house PCR assay targeting the nuc, mecA, and mecC genes [27].

Definition of S. aureus Nasal Carriage Status
Samples with discrepant results between the BD MAX StaphSR assay and the culture methods were tested with the Xpert SA Nasal Complete assay (Cepheid, Maurens-Scopont, France).Samples were considered positive for S. aureus or MRSA if at least the cultures or the Xpert SA Nasal Complete assay detected S. aureus or MRSA, respectively.The results of direct and pre-enriched cultures combined to the result of the Xpert SA Nasal Complete assay were considered as the gold standard status.Statistical analyses were performed using the MedCalc Statistical Software version 22.007 (MedCalc Software Ltd., Ostend, Belgium).

Conclusions
To conclude, the BD MAX StaphSR assay is a fully automated third-generation NAAT that enables rapid and accurate detection of S. aureus and MRSA nasal carriers and could help to reduce both nosocomial and endogenous infections by S. aureus.

Table 1 .
Performance of the BD MAX StaphSR assay for the detection of S. aureus according to the sample status defined by different techniques.

Table 2 .
Performance of the BD MAX StaphSR assay for the detection of methicillin-resistant S. aureus (MRSA) according to the sample status defined by different techniques. 1 NPV: Negative predictive value. 2 PPV: Positive predictive value.

Table A2 .
Results of samples showing discrepant results (n = 52) with the BD MAX StaphSR assay, the direct and pre-enriched culture, and the Xpert SA Nasal Complete assay.